Bacillus anthracis is the causative agent of the anthrax disease, a rare disease in animals and even more rare in humans. However, because it forms stable spores that can be sprayed in the air, B. anthracis is a potential biological warfare and bioterrorism agent. The accidental release of spores into the air in Sverdlosk, Russia, and the recent mail attack in the United States in the Fall 2001 led to many deaths, and sadly demonstrated the infectivity and threat of the bacterium. Anthrax toxin is the major virulence factor of B. anthracis and consists of three proteins: lethal factor (LF), edema factor (EF), and protective antigen (PA). We are investigating the protein translocation pathways across the cell wall envelope of B. anthracis as potential targets for antimicrobial therapy. Experiments described in this proposal aim at identifying B. anthracis genes that are required for the secretion of PA, LF and EF across the cell wall envelope of the bacterium. By conducting a generalized transposon mutagenesis and scoring for defects in the secretion of exotoxins, we will ask whether we can identify all genes required for B. anthracis secretion. The identified genes will be examined for their role in B. anthracis virulence. Isogenic knockout variants (at least 10 different variants) will be generated in B. anthracis strain Ames and subjected to virulence studies in the guinea pig infection model. In addition, knockout mutations in the three prsA-like genes of B. anthracis will be generated, and we will ask whether the encoded peptidyl-prolyl isomerases are essential for the secretion of the toxins as reported for B. subtilis. If we can identify genes and gene products in the B. anthracis secretory pathway that are required for virulence, the gene product could represent a novel target for antimicrobial therapy.